KR100219256B1 - Novel polypeptides with affinity to lipopolysaccharides and their uses - Google Patents

Abstract

A novel polypeptide sequence having the formula <IMAGE> in which A1 is a hydrogen or at least one and no more than two amino acids selected from the group consisting of lysine and arginine, A2 is a tyrosine, phenylalanine or tryptophan residue, A3 is an arginine or lysine residue, A4 is at least one and no more than two amino acids selected from the group consisting of lysine and arginine, and A5 is an -OH or an NH2, is described. The polypeptide may be used in a pharmaceutical composition as an antimicrobial or antiviral agent, specifically as an anti-HIV agent. <IMAGE>

Description

[Name of invention]

New polypeptides having affinity for lipopolysaccharides and uses thereof

[Field of Invention]

The present invention relates to novel polypeptide (s) or pharmaceutically acceptable salts thereof which exhibit a strong affinity for lipopolysaccharides, in particular endotoxin. This polypeptide can be used as an anti-bacterial or anti-viral agent (eg, HIV agent) in pharmaceutical crude.

[Background of invention]

Two families of antimicrobial polypeptides have been isolated from crabs (eg Shigenaga, 1990. J. Biol. Chem. 265: 21350-21354; Kawano et al., 1990, J. Biol. Chem. 265: 15365-15367). Muta et al., 1990, J. Biochem. 108: 261-266; Japanese Patent Laid-Open No. 167230/1990; Japanese Patent Laid-Open No. 152987/1990; Japanese Patent Laid-Open No. 53799/1990; Published searched Application 500194 / 1990; Miyata et al., 1989, J. Biochem. 106: 663-668; Akaji et al., 1989, Chem. Pharm. Bull. 37: 2661-2664; Tainome 26: 301-311 (1989); Shieh et al., 1989, FEBS Lett. 252: 121-124; and Nakamura et al., 1988, J. Biol. Chem. 263: 16709-16713). One of them, the tachyplesin family, was isolated from the Japanese crab, Tachpleus.

Three tachiflesin, I, II and III were identified; Their amino acid sequence is shown in FIG. In addition, tachyflecin peptide derivatives having extended carboxyl ends with glycine-lysine have been found in Southeast Asian crab species, Carcinoscorpius rotundicauda (Muta et al., 1990, J. Biochem. 108: 261-). 266). The second family, Polyfemucin family, was isolated from the blood cells of US crab, Limulus polyphemus. Two polyfemucins, I and II, have been identified; Their amino acid sequence is shown in FIG. Both families of polypeptides consist of 17 or 18 amino acid residues and typically have four conserved regions and two disulfide bridges (see Figure 1). Both tachiflesin and polyfemucin have been found to inhibit the growth of gram-negative and positive-positive bacteria at low concentrations as well as fungi such as Candida albicans and to form complexes with bacterial lipopolysaccharides (Shigenaga 1990, J. Biol. Chem. 265: 21350-21354 and Muta et al., 1990, J. Biochem. 108: 261-266).

Polypeptides with tachiflesin also include influenza viruses, bullous stomatitis virus (Murakami et al., 1991, Chemotherapy, 37, 327-334) or human immunodeficiency virus (Moritomo et al., 1991, Chemotherapy, 37, 206-211). It was found to exhibit some inhibitory activity against the virus.

[Overview of invention]

The present invention relates to novel polypeptide (s) derived from, and with some similarity to, endotoxin high affinity polypeptides of crab, especially tachiflesin and polyfemucin. In a polypeptide polypeptide, the amino acid at position 6 of tachiflesin or the amino acid at position 7 of polyfemucin is valine, ie a neutral aliphatic amino acid. In the polypeptide (s) of the invention, the amino acid residue at position 6 is a lysine (Lys) or arginine (Arg) residue; These are basic amino acid residues having properties that are substantially different from valine residues. Further, the amino acid residue at the 11-position in the polypeptide of the present invention is tyrosine (Tyr), phenyl alinine (Phe) or tryptophan (Trp), ie, having different properties from the 11-position of isoleucine (Ile) of Tachiflesin. Aromatic amino acid residues.

The novel polypeptides of the invention can be used as anti-HIV agents. As detailed in section 6 below, the polypeptide (s) of the invention have significantly higher anti-HIV activity compared to known endotoxin high affinity polypeptides of crab.

[Justice]

Peptide sequences as defined herein are represented by three letter abbreviations for the following amino acid residues:

Ala (alanine); Arg (arginine); Cys (cysteine); Gly (glycine); Ile (isoleucine); Leu (leucine); Lys; Phe (phenylalanine); Trp (tryptophan); Tyr (tyrosine); And Val (valine).

The following terms as used herein will have the meaning as indicated.

HIV = human immunodeficiency virus (all variants)

MOI = multiplicity of infection

SI = selectivity index (ratio of CC ₅₀ to EC ₅₀ )

[Brief Description of Drawings]

1 shows the amino acid sequence of Tachiflesin I, Tachiflesin II, Tachiflesin III, Polyfemucin I, and Polyfemucin II. Conserved amino acids are in boxes. Disulfide bonds between Cys-3 or -4 and Cys-16 or -17 and between Cys-7 or -8 and -12 are indicated by solid lines.

2 shows a synthetic diagram for synthesizing the polypeptide (1) of the present invention.

Detailed description of the invention

The present invention relates to novel polypeptides of the formula (I) or salts thereof.

[Wherein A ₁ is at least one and up to two amino acids selected from the group consisting of hydrogen or lysine and arginine, A ₂ is a tyrosine, phenylalanine or tryptophan residue, A ₃ is an arginine or lysine residue, A ₄ is at least one and up to two amino acids selected from the group consisting of lysine and arginine, A ₅ is -OH (derived from carboxyl groups) or -NH ₂ (derived from acid amide groups), and Cys is cysteine Residue, and Gly is a glycine residue.]

In certain embodiments, cysteine residues in 3- and 16-positions and / or cysteine residues in 7- and 12-positions may be linked via disulfide bonds (-S-S).

Specific examples of the polypeptide of the present invention represented by structural formula (I) are shown in Table 1 and numbered as (1) to (33). Each symbol represents a corresponding amino acid residue by an internationally recognized three letter representation (see section 3.1 above).

Similar to the endotoxin high affinity polypeptides isolated from sesame known in the art, the polypeptides of the invention are antiparallel beta due to intermolecular hydrogen bonding and the presence of four cysteine residues at the 3-, 7-, 12- and 16-positions. It has an antiparallel beta-sheet structure. Perhaps the turning positions of the beta-trun structures are in the 9- and 10-positions. The peptide portion at the 3-position to 8-position and the peptide portion at the 11-position to 16-position face each other.

However, in contrast to known polypeptides isolated from crab, the polypeptide (s) of the invention are more basic. In particular, the amino acid residue at the 6-position is an arginine (Arg) or lysine (Lys) residue. The amino acid residue at position 6 in the polypeptide isolated from crab is a valine residue.

Due to their basic properties, the polypeptide (s) of the invention can form salts by acid addition. For example, the polypeptide may be an inorganic acid (hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), or an organic carboxylic acid (acetic acid, halo acetic acid such as trifluoroacetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid). Salts such as salicylic acid and uronic acid such as glucuronic acid or hyaluronic acid) or sulfonic acid such as chondroitin sulphate (methanesulfonic acid, p-toluenesulfonic acid, etc.).

[Production of Polypeptide]

The novel polypeptides of the present invention can be prepared by methods known in the art, such as solid phase synthesis described by Solid-Phase Peptide Synthesis, Stewart Young, Pierce Chemical company, Rockford, Illinois (1984). That is, the straight chain polypeptide of the present invention of formula (I) is a spacer having a functional group capable of binding a carboxyl group to an insoluble resin having an amino group attached directly or alternately attached to the carboxyl group of the N-protected arginine at the 17-position. (For example, one capable of converting a carboxyl group of arginine to a p-carboxybenzyl ester). The amino group of an insoluble resin having an argining (Arg) residue at the 17-position after deprotection of the -amino (Na) -protecting group is in the 16- to 1-position of the amino acid sequence of the structural formula (I) It can be linked in series with each protective amino acid, after which the protecting group of the insoluble resin and amino acid is removed.

[SEQ ID NO 1]

[Wherein A ₁ , A ₂ , A ₃ , A ₄ , A ₅ , Cys and Gly are as defined in the above formula (I).]

In this case, the carboxyl terminus of the amino acid residue at the 17-position may be in the free state (A ₅ corresponds to -OH) or converted to the acid amide form (A ₅ corresponds to -NH ₂ ). Two cysteines in the 3- and 16- and 7- and 12-positions can form disulfide bonds (-SS) through mercapto groups by air oxidation, or disulfide bonds are described in Atherton, E. et al .; J. Chem. Soc., Perkin Trans. Selectively protecting the mercapto group of one of the cysteine group pairs in 3- and 16- and 7- and 12-positions with the protecting group, t-Bus (t-butylthio), according to the method of 1, 1985, 2065, Protecting the remaining cycapto group mercapto group with a protecting group, Acm (acetamidomethyl); remove t-Bus and partially oxidize the mercapto group; The step of removing the Acm protecting group can be formed through a step that is performed using procedures known in the art.

An insoluble resin having an amino group is an insoluble resin having any amino group as long as it can be removed after being linked to the carboxyl group of the C-terminal N-protected arginine or lysine or, in certain cases, to the carboxyl group of the spacer bound thereto, via its amino group. It can be used to synthesize novel polypeptides of the invention. Examples of such insoluble resins include, but are not limited to, amino-methyl resins (aminomethylated styrene-divinylbenzene copolymers), benzhydrylamine resins, methylbenzhydrylamine resins and aminomethylphenoxymethyl resins and derivatives thereof. It doesn't happen. When benzhydrylamine resin, methylbenzhydrylamine resin, dimethoxybenzhydrylamine (DMBHA) resin or aminomethylphenoxymethyl resin is used, the amide is directly obtained by decomposition, but the amino-methyl resin is yield Preferred at

Each amino acid that can be used in the solid phase synthesis method may be L-type or D-type. A protective amino acid may be an amino acid whose functional group may be protected using a method known in the art, or may be a variety of commercially available protective amino acids. Those skilled in the art will recognize that polypeptide synthesis methods require the use of protecting groups to stabilize the destabilizing side chains of amino acids to prevent chemical modification of the amino acid side chains during the synthesis process. Amino acid Protecting groups for amino groups include, but are not limited to, groups selected from Boc (t-butyloxycarbonyl) or Fmoc (9-fluorenylmethyloxycarbonyl). The protecting group for the guanidino group of arginine (Arg) is Tos (tosyl), NO ₂ (nitro), Mtr (4-methoxy-2,3,6-trimethylbenzene-sulfonyl) or Pmc (2,2,5) , 7,8-pentamethyl-cropan-6-sulfonyl), but is not limited thereto. The protecting groups for the mercapto group of cysteine (Cys) are Bz1 (benzyl), MBz1 (4-methoxybenzyl), 4-MeBzl (methylbenzyl), Acm (acetamidomethyl), Trt (trityl), Npys (3 -Nitro-2-pyridinesulphenyl), t-Bu (t-butyl) or t-Bus (t-butylthio) and Mbzl, but are not limited to 4-MeBzl, Trt, Acm and Npys is preferred. The protecting group for the hydroxyl group of tyrosine (Tyr) includes, but is not limited to, a group selected from Bzl, Cl ₂ Bzl (2,6-dichlorobenzyl) or t-Bu. Lysine The protecting group for an amino group is a group selected from Z (benzyloxycarbonyl), C1Z (2-chloro-benzyloxycarbonyl), Boc or Npys, but is not limited thereto.

Coupling of protective amino acids can be carried out by condensation methods known in the art, such as the DCC (dicyclohexylcarbodiimide) method, DIPCDI (diisopropylcarbodiimide) method [Tartar, A. et al., 1979, J. Org, Chem. 44: 5000], active ester method, mixed or symmetric acid anhydride method, carbonyldiimidazole method, DCC-HOBt (1-hydroxybenzotriazole) method [Konig W. et al., 1970, Chem. Ber., 103: 788, 2024, 2034 or the diphenylphosphoryl azide method, but preferably the DCC method, the DCC-HOBt method, the DIPCDI-HOBt method or the symmetric acid anhydride method is used. The condensation method can be carried out in an organic solvent such as dichloromethane or dimethylformamide, or a mixed solvent thereof. Deprotectants such as trifluoroacetic acid / dichloromethane, HIC / dioxane, piperidine / dimethylformamide (DMF) Used to deprotect the protecting group for an amino group. The progress of the condensation reaction at each stage of the synthesis was determined by the method of E. Kaiser et al. [Anal. Biochem., 34, 595 (1970)] (ninhydrin reaction method).

When aminomethyl resin derivatives are used as insoluble resins, protective polypeptides can be removed from the resin by treating the protective peptide resin with ammonia in a suitable solvent. The resulting protective peptide is treated with hydrogen fluoride to give a polypeptide amide represented by the above structural formula and free of protecting groups. Benzhydrylamine resins, methylbenzhydrylamine resins, aminomethylphenoxymethyl resins or DMBHA resins [Funakoshi, S. et al .; J. Chem. Soc., Chem. When Commun., 1988, 382] is used as an insoluble resin, the polypeptide may be removed from the resin and the protecting group may be selected from the group consisting of a protective peptide resin, hydrogen fluoride, TFMSA (trifluoromethanesulfonic acid), published by Academic Press, E. Gross; Yajima, H and the like; "The Peptides" vol. 5, Page 65 (1983)], TMSOTf (trimethylsilyl triflate) [trimethylsilyl bromide] [Fujii, N, et al .; Chem. Pharm. Bull., 35, 3880 (1987), and the like.

In a preferred embodiment, the resulting polypeptide is reduced to 2-mercaptoethanol, DTT (dithiothritol) or the like so that the mercapto group of cysteine is in reduced form. The mercapto group can then be oxidized to yield a cyclic polypeptide. The oxidation treatment can be carried out by a known method. Typically oxidants such as air or ferricyanate (eg potassium ferricyanide) are used.

Alternatively, polypeptide (s) of the invention can be prepared using recombinant NDA techniques. Thus, nucleotide coding sequences for polypeptide (s) of the invention can be cleaned and expressed using techniques known in the art. See, eg, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1991 by Maniatis et al.

Polypeptides of the present invention can be isolated and purified by known techniques in the art, such as extraction, recrystallization, various chromatography (gel filtration, ion exchange, partitioning, adsorption, reverse phase), electrophoresis, countercurrent distribution, and the like. Reverse-phase high performance liquid chromatography is most effective.

[Use of Polypeptides]

The polypeptide (s) of the present invention represented by structural formula (I) have the ability to bind endotoxins, antibacterial activity and hemolytic endotoxin-sensitized blood cells. In addition, the polypeptide (s) of the invention have antiviral activity. In certain embodiments, polypeptide (s) of the invention have anti-HIV activity. As described in detail in section 6 below, the polypeptides of the present invention have significantly higher anti-HIV activity than known endotoxin high affinity polypeptides (e.g., tachiflesin I, II or III or polyfemucin I or II). Indicates.

Therefore, the polypeptide (s) of the present invention can be used in pharmaceutical compositions comprising the polypeptide (s) of the present invention or salts thereof and a pharmaceutically acceptable carrier selected according to the method and dosage form of the pharmaceutical composition. Pharmaceutical carriers may be physiologically compatible carriers such as Hank's or Ringer's solutions, mixtures of physiological saline, saline and glucose, and sodium heparinated-citrate-citric acid-textrose solutions. Pharmaceutical compositions are administered orally or parenterally, depending on the subject of treatment, and as powders, granules, injections or oral dosages, tablets, suppositories, pessaries, ointments, creams or aerosols, using appropriate pharmaceutical carriers depending on the method of administration Can be prepared.

When the pharmaceutical composition is administered directly by injection to a patient, the polypeptide of the present invention or a salt thereof is continuously administered by intravenous infusion as a physiological saline solution in an amount of 10 to 5.000 mg / kg body weight per day. Or intermittently.

EXAMPLE

In this example, the synthesis of the polypeptide (1) is described. In addition, results of anti-HIV activity assays of polypeptides of the invention and known endotoxin high affinity polypeptides are disclosed. Polypeptides of the invention have significantly higher anti-HIV activity compared to known endotoxin high affinity polypeptides.

In the examples, the following instruments and reagents are used:

HPLC instrument: Waters co. (USA) Model 600

Column of the instrument: Asahipak ODP-90 (Asahi Chemical Industry Co., Ltd.)

Fmoc Amino Acid: Kokusan Kagaku co., Ltd. Produce

Amino Resin and Condensing Agent: Peptide Kenkyusho Co., Ltd. Produce

FAB-MS (FAB-Mass Spectrophotometer): VC Co. (USA), Model ZAB-SE

Example 1 Synthesis of Polypeptide (1)

Synthesis of polypeptide (1) having the structural formula below is described in sections 6.1.1 to 6.1.7 below. Polypeptide 2-33 (see Table 1 for structure) is synthesized using a similar method.

Cysteine residues in the 3- and 16- and 7- and 12-positions are each linked via disulfide bonds.

[Fmoc-DMBHA-CH ₂ CH ₂ COOH [(3- ( -Fmoc-amino-4-methoxybenzyl) -4-methoxyphenyl) propionic acid] to aminomethyl resin]

270mg (0.2mmole) of aminomethyl resin (0.74meq / g) and 268.5mg (0.5mmole, 2.5eq) of Fmoc-DMBHA-CH ₂ CH ₂ -COOH (MW 537) were added to a solid-phase synthesis column and condensation reaction was performed. , L. et al. ((Chem. Pharm. Bull., 36, 4989 (1988)) according to the method of DIPCDI-HOBt in DMF for 2 hours.

After completion of the condensation reaction, the coupling is carried out using acetic anhydride (DMBHA resin) for the protection of free amino groups.

[Introduction of 17-Position Arginine into DMBHA Resin]

Fmoc group 20 from DMBHA prepared in section 6.1.1 above After removal with piperidine / DMF, 2.5 equivalents (eq) of Fmoc Arg (Mtr) -OH are added, based on the DMBHA resin, and the condensation reaction is carried out in DMF according to the DIPCDI-HOBt method.

The progress of the condensation reaction was determined by Kaiser, E. et al. [Anal. Biochem., 34, 595 (1970)] in accordance with the ninhydrin test.

[Introduction of 16-position Cysteine]

Fmoc groups from DMBHA resin After removal with piperidine / DMF, 2.5 eq of Fmoc-Cys (MBzl) -OH based on DMBHA resin is added and the condensation reaction is carried out in DMF by the DIPCDI-HOBt method. The progress of the condensation reaction is measured according to the ninhydrin test similar to 6.1.2 above.

[6.1.4 Introduction of Amino Acids from 15-position to 1-position]

Similar to the above, Lys (Boc), Arg (Mtr), Tyr (t-Bu), Cys (MBzl), Tyr (t-Bu), Gly, Lys (Boc), Tyr (t-Bu), Cys (MBzl), Lys (Boc), Arg (Mtr), Tyr (t-Bu), Cys (MBzl), Trp, Arg (Mtr) and Arg (Mtr) were subsequently introduced into the DMBHA resin to protect with a protecting group (1) Obtain a resin.

Each amino acid condensation reaction in the solid phase synthesis is carried out according to the operating conditions of Table 2.

6.1.5 Preparation of Polypeptide (1) by Removal of Protecting Group, Removal of Polypeptide (1) from Resin and Partial Purification]

Protective polypeptide (1) resin 20 Piperidine / DMF treatment to remove Fmoc groups and then 10 ml of trifluoro in the presence of 1M TMSOTf-thioanisole / TFA (trifluoroacetic acid) based (m-cresol (100eq) and ethanedithiol (300eq) per 100 mg of resin In acetic acid) The reaction is carried out for 2 hours. The resin is filtered from the reaction mixture and washed twice with 1 ml of trifluoroacetic acid. 100 ml of ice-cold dry ether is added continuously to the mixture of filtrate and washings. The precipitate formed is centrifuged and the residue is separated from the supernatant by decantation. The resulting residue is washed with cold ether, dissolved in 10 ml of 4N AcOH, 830 mg, 80 eq dithiothreitol are added and the mixture is stirred at rt overnight.

The reaction solution was centrifuged, the supernatant was treated with Sephadex G-10 (3.7 × 5 cm), gel filtered with 4N acetic acid (AcOH), the flow-through was collected as the main elution and the powder was lyophilized. To obtain partially purified non-cyclic polypeptide (1).

6.6.1 Preparation of Polypeptide (1) by Air Oxidation

The cyclization reaction is carried out by adjusting the pH of the 1/2 amount flow-through fraction to 7.5 with concentrated aqueous ammonia and air oxidizing by aeration. After completion of air oxidation, the cyclized polypeptide (1) was adsorbed onto 10 g of Diaion HP-20 resin, followed by 60 Elute with a CH ₃ weight (in 1N AcOH). The eluate is concentrated under reduced pressure at room temperature to remove CH ₃ CN and then lyophilized to give a powder. The powder was dissolved in a small amount of water, the solution was poured into Asahipak and purified by high performance liquid chromatography (HPLC-Model 600 from Waters Co.) with gradient elution with CH ₃ CN to yield 27 (The value calculated on the basis of the protecting group-protecting polypeptide (1) resin) yields a single peak of polypeptide (1).

6.7.1 Analysis of Polypeptides

When the amino acid composition was measured by leucine aminopeptidase digestion of the polypeptide purified in Section 6.1.6 above, it can be seen that it is within the range of composition calculations based on the amino acid sequence of formula (1).

Intrinsic Light Rotation of the Obtained Polypeptides Is + 8.4 °) C = 0.1, 1N acetic acid).

6.2 Example 2: Antiviral Activity Against Human Immunodeficiency Virus (HIV)

Polypeptides (1) synthesized in Example 1 as well as polypeptides (2), (3), (4), (7), (12), (13), (14), (20), (21) and ( Antiviral activity against HIV of 26) is tested and evaluated according to the following method.

HIV-infected MT-4 cells (2.5 × 10 ⁴ cells / well, multiplicity of infection (MOI): 0.001) are added to 96-well microtitre plates with varying concentrations of test substance immediately after infection. 37 After 5 days incubation in a CO ₂ incubator, the number of viable cells was determined by MTT method [Pauwels et al .; J. Virol. Methods, 20, 309 to 321 (1988)]. Antiviral Activity 50 Cell Death Due to HIV Infection Inhibitory concentration (EC ₅₀ : 50 Effective concentration). On the other hand, in order to know the cytotoxicity of the test substance against MT-4 cells, virus-non-infected cells are incubated with test compounds of various concentrations as described above. Cytotoxicity Caused by Test Substances 50 Shown as cytotoxic concentration (EC ₅₀ ). Also it shows the CC ₅₀ dae Love (rough) ratio of the _{EC 50 (CC 50 / EC 50} ) as an effective ratio (SI = selectivity index).

Table 3 lists endotoxin covalents with polypeptides (1), (2), (3), (4), (7), (12), (13), (14), (20), (21), and (26). EC ₅₀ , CC _50, and SI values of known polypeptides having Mars, in particular, Tachiflesin I and II and polyfemucin I and II and known anti-HIV agent AZT.

Table 4 shows the physical property values of the polypeptides (1), (3), (13), (20) and (21).

The results shown in Table 3 show that the polypeptides synthesized in Example I have significantly high anti-HIV activity when measured at effective concentrations. Compounds (1), (3), (12), (13), (14) and (26) are particularly selective for killing HIV infected cells. The selectivity index of compounds (1), (3), (12), (13), (14) and (26) is at least 7 times higher than any known endotoxin high affinity compound.

Since the implementations disclosed herein are intended to describe some areas of the invention, the inventions described and claimed herein are not limited by these specific embodiments. Any equivalent embodiment is within the scope of the present invention. Various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications will also fall within the scope of the appended claims.

Various references are hereby incorporated by reference in their entirety.

Claims (11)

Polypeptides or salts thereof [Wherein A ₁ is at least one and up to two amino acids selected from the group consisting of hydrogen or lysine and arginine, A ₂ is a tyrosine, phenylalanine or tryptophan residue, A ₃ is an arginine or lysine residue, A ₄ is at least one and up to two amino acids selected from the group consisting of lysine and arginine, 9A ₅ is —OH or HN ₂ , Cys is a cysteine residue and Gly is a glycine residue.] The polypeptide or salt thereof of claim 1, wherein A ₁ is hydrogen. The polypeptide or salt thereof according to claim 1, which is at least one amino acid selected from the group consisting of lysine and arginine of A ₁ . The polypeptide or salt thereof of claim 1, wherein A ₁ is two amino acids selected from the group consisting of lysine and arginine. The polypeptide or salt thereof of claim 1, wherein A ₄ is at least one amino acid selected from the group consisting of lysine and arginine. The polypeptide or salt thereof of claim 1, wherein A ₄ is two amino acids selected from the group consisting of lysine and arginine. The polypeptide or salt thereof of claim 1, wherein the cysteine residues at the 3- and 16-positions are linked via disulfide bonds. The polypeptide or salt thereof of claim 1, wherein the cysteine residues at the 7- and 12-positions are linked via disulfide bonds. Polypeptides or salts thereof of the formula: [Wherein A ₁ is at least one and up to two amino acids selected from the group consisting of hydrogen or lysine and arginine, A ₂ is a tyrosine, phenylalanine or tryptophan residue, A ₃ is an arginine or lysine residue, A ₄ is at least one and up to two amino acids selected from the group consisting of lysine and arginine, A ₅ is —OH or —NH ₂ , Cys is a cysteine residue, Gly is a glycine residue; Cysteine residues in 3- and 16-positions are joined via disulfide bonds, and cysteine residues in 7- and 12-positions are joined via disulfide bonds.] A pharmaceutical composition for inhibiting HIV activity in a patient comprising an effective amount of the polypeptide of claim 1 and a pharmaceutical carrier. A pharmaceutical composition for inhibiting HIV activity in a patient comprising an effective amount of the novel polypeptide of claim 9 and a pharmaceutical carrier.